Sedimentation Velocity Sample Guide

Sedimentation Velocity

Sample Preparation Guide

Good results can only be obtained with well prepared sample and adequate data collection. In order to assess the concentration dependence of the sedimentation coefficient, it is necessary to collect data using three protein concentrations.
- Normally, the three initial concentrations would be at starting optical densities of 0.8, 0.5, 0.3 at the desired wavelength (anywhere from 225 to 800 nm). The velocity experiment requires significantly more sample than the equilibrium experiment, and you will need at least 1 ml of sample at the highest concentration along with 2 mls of buffer. Sample dilution will be done when the experiment is set up. Excess sample will be cared for and returned to you. Additionaly, most samples can also be recovered at the end of the experiment, if so desired.
The sample should be dialyzed to equilibrium against the buffer. During the experiment, the buffer absorbance will be automatically subtracted from the sample absorbance. Any optical imbalance in these two samples (besides the protein) will contribute to the absorbance versus radius profile. Salt imbalances can give rise to Schlieren effects.
Some comments on buffer choice: Your buffer solution should contain 20 mM buffering ions with 150-200 mM salt for ionic strength. A typical buffer would be sodium phosphate, pH 7.0, 200 mM NaCl. Tris buffer can also be used as long as the sample will be monitored at 280 nm. Tris buffer interferes with protein detection at 230 nm. If a reducing agent is required, beta-mercaptoethanol is preferred over dithiothreitol since it does not have any absorbance at 280 nm.
Indicate the temperature range at which the sample is stable. The standard procedure is to conduct the velocity experiment at 20 degrees C so that a temperature correction will not be necessary. Depending on the size of your protein, the experiment can take anywhere from 2 to 5 hours to collect. It is absolutely essential that the protein sample remain stable throughout the duration of the experiment. The analytical ultracentrifuge is equipped with a temperature controller such that data can be collected within a range of 2 to 40 degrees C.